Plasmodium falciparum merozoite proteins have been considered as a major target for developing a subunit vaccine that could block the malaria parasite invasion of erythrocytes. For most merozoite antigens, however, their molecular function during the invasion process is not well understood. Recently, we have discovered that two non-glycosylated ectoplasmic regions of band 3 function as a crucial erythrocyte receptor and interact with a number of P. falciparum merozoite proteins by a sialic acid-independent mechanism. We were able to identify one of these proteins as merozoite surface protein-1 (MSP1). However, other merozoite proteins binding to the band 3 receptor could not been identified by biochemical methods. Based on our findings, we hypothesize that band 3 is a crucial host receptor interacting with multiple parasite ligands in sialic acid-independent manner during the P. falciparum invasion of erythrocytes. The long-term objective of our study is to identify target antigens for an effective malaria vaccine on the basis of well-defined functions of the antigens. In this study, we propose to test our hypothesis by identifying the merozoite antigens that bind to the band 3 receptor. The specific aims of the study are as follows. (a) Screening of P. falciparum cDNA library: Using the receptor region of human band 3 as the bait, parasite proteins binding to the band 3 receptor will be identified by screening a cDNA library in a yeast two-hybrid system in vivo. (b) Independent confirmation of the interactions: The binding of newly identified parasite ligands to band 3 will be confirmed in solution in vitro using quantitative methods. (c) Production and characterization of antibodies to the identified parasite ligands: Polyclonal antibodies will be generated in rabbits against recombinant proteins and characterized using native parasite proteins. (d) Localization of the parasite ligands to merozoites: Indirect immunofluorescence microscopy will be carried out using the produced antibodies and the parasites at the segmented schizont stage to localize the band 3-binding parasite antigens to the merozoite. We anticipate this study will make a significant contribution towards the identification of potential new targets for a malaria vaccine and provide a better understanding of the sialic acid-independent erythrocyte invasion mechanism in P. falciparum malaria. [unreadable] [unreadable]